Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s13577-024-01054-1.


Introduction
Cutaneous squamous cell carcinoma (cSCC) is a prevalent type of nonmelanoma skin cancer, accounting for 20% of all skin cancer cases [1].Its incidence is increasing in most white populations globally [1].Depending on the origin of the tumor, cSCC can be classified as cutaneous or invasive.Cutaneous cSCCs arise from keratinocyte proliferation, while invasive cSCCs arise from intraepidermal dysplasia [2].The primary risk factors for cSCC development include exposure to ultraviolet radiation, chemical carcinogens, immunodeficiency, chronic inflammation, and precursor pre-malignant conditions, including Bowen's disease, actinic keratosis, and keratoacanthomas [3][4][5].
The European guideline categorizes cSCCs as primary or advanced, depending on the extent of tumor spread, and primary cSCCs are typically non-metastatic and easily treatable, whereas advanced cSCCs are classified as locally advanced or metastatic.In the early stages of development, cSCCs tend to be indolent with low metastasis rates, resulting in a better prognosis with 5-year cure rates greater than 90% [6].For small tumors (≤ 1 cm in diameter) with defined primary lesions, effective treatments such as electrodesiccation, curettage, excision, or cryosurgery have shown up to 90% success rates [5].However, tumors with high metastatic risk and potential aggressiveness require prophylactic irradiation of the regional lymph nodes after surgery, as they become highly aggressive when recurring (8).Advanced cSCCs have a worse prognosis, with 10-year survival rates of less than 20% for patients with regional lymph and less than 10% for those with distant metastases, while approximately 90% of patients with distant metastases had died due to the disease within five years [7,8].
Understanding the complex molecular profile of cSCC is crucial for developing novel and effective therapies, particularly for the rare metastatic cSCC [9].In vitro and in vivo cellular models are essential tools for discovering better treatments.However, the limited availability of wellcharacterized cSCC lines for preclinical experimentation and drug screening presents a challenge [11].Currently, the A431 cell line derived from the vulva [12] is the only commercially available cSCC line in the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) cell banks.Recently, a patientderived cutaneous squamous cell carcinoma (cSCC) xenograft (PDX) was generated, replicating both the histological and genetic attributes of the primary tumor, and exhibiting a response profile to eribulin and cisplatin, underscoring the critical utility of establishing primary lineages for delineating novel therapeutic strategies [10].
Therefore, this study aimed to establish and characterize a new advanced cSCC cell line that preserves the biological properties of metastatic cancer cells and can be used for future investigations of their biology and novel effective anti-cancer agents.

Clinical presentation
A 67-year-old male patient had undergone excision of cutaneous epidermoid carcinoma on his anterior chest the year before and sought medical care at the surgical oncology outpatient clinic at Barretos Cancer Hospital, Brazil, with a persistent tumor in the left armpit (Supplementary Fig. 1).The patient had a history of acute myocardial infarction, coronary stent placement, and diabetes.A tumor biopsy at Barretos Cancer Hospital revealed a cutaneous squamous cell carcinoma (cSCC), and the patient underwent surgery and received postoperative carboplatin, taxane, and radiation therapy.Unfortunately, the patient experienced a recurrence shortly after treatment and was deemed no longer treatable, ultimately resulting in his death.

HCB-541 cell line establishment
Surgical specimens were collected for cell cultivation immediately after surgery and maintained in Dulbecco's Phosphate Buffered Saline solution until processing begins as previously reported [13].Biopsy tumor was fragmented into small pieces with a surgical scalpel in a petri dish.Then, it was incubated in 0.5 ml of accutase/trypsin enzymatic solution for total tissue dissociation, at 37 °C for 30 min.After dissociation, the cell solution was incubated in DMEM medium supplemented with 10% Fetal Bovine Serum (SBF) and 1% penicillin and streptomycin to complete enzyme inactivation.Throughout the study, experiments were consistently conducted using aliquots from this identical FBS batch (210625 K) to ensure quality and minimize variations in component composition.Cells were cultivated in DMEM medium supplemented with 1% penicillin/streptomycin, 10% FBS and maintained in culture flask at 37 °C, 5% CO 2 .Primary cell line was named HCB-541, and stock vials were frozen in different passage numbers to report the protein expression change during establishment processes, and for DNA and RNA analysis at the 15th passage.

Morphological and doubling time characterization
To assess the morphologic profile, after continuous passages (> 30), the HCB-541 cell line was seeded in plates and cultivated in DMEM supplement with 1% P/S and 10% SFB at 37 °C, 5% CO2.Cells were photographed under an optical microscope at × 100 magnification (Olympus XT01), until complete 90% of total confluence.Doubling time values were obtained using xCELLigence real-time cell analysis (Agilent Technologies, Inc), according to the manufacturer's instructions.Initially, 3.5 × 10 3 or 7 × 10 3 cells were plated into E-plate (Agilent Technologies, Inc) and were cultivated in DMEM medium supplemented with 1% P/S and 10% for 72 h.The cell index was calculated using a specific RCCA® software analysis.

Tumorigenicity of HCB-541 cell line in NOG-SCID mice
To assess the tumorigenicity of HCB-541, subcutaneous injections of either 1 × 10 6 or 3 × 10 6 cells mixed with Matrigel® at a 1:1 (v/v) ratio were administered into the right flank of 8-week-old female NSG mice (NOD.Cg-Prkd cscid Il2rg tm1Wjl /SzJ, The Jackson Laboratory, USA), as previously described [14].Tumor size was measured weekly with calypter, and volume was calculated using the formula: volume = length × width2/2.After two weeks of transplantation, tumors were excised surgically, measured, and subjected to morphological evaluation using hematoxylin and eosin staining and immnunohistochemistry (IHC) analysis.Tumors were fixed in a 4% paraformaldehyde, and paraffinembedded sections were prepared using standard procedures for histological analysis.The mice used in this study were maintained under SPF conditions, on a 12-h light/dark cycle, and received sterile food and water ad libitum.

Cell line authentication by short tandem repeat (STR) profiling analysis and mycoplasma contamination test
STR analysis was performed using DNA from the cell line and FFPE tumor biopsy as previously described for authenticity confirmation [15].The mycoplasma contamination test was carried out in DNA from the cell line using MycoAlert Mycoplasma Detection Kit (Lonza, Swiss), according to the manufacturer's protocol, in different moments throughout the time it was in culture.

DNA and RNA isolation
DNA was isolated from the patient's formalin-fixed and paraffin-embedded (FFPE) tumor biopsy, using QIAamp DNA Micro Kit (Qiagen) as previously reported [16].DNA from the HCB-541 cell line was isolated using Biopur Mini Spin Plus 250 Extraction kit (Biopur), both according to the manufacturer's recommendations.All samples were quantified by NanoDrop 2000 System (Thermo Scientific) and stored at −20 °C.RNA was isolated from HCB-541 and HACAT (immortalized normal human keratinocyte) cells using RecoverAll Total Nucleic Acid Isolation kit (Invitrogen), according to the manufacturer's recommendations.Samples were quantified by NanoDrop 2000 System (Thermo Scientific) and Qubit 2.0 Fluorometer (Life Technologies), and stored at −80 °C.

Cellular viability assay (MTS)
Cell viability was determined 72 h after drug exposure, using the colorimetric CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI), according to the manufacturer's instructions and as previously reported [15].To assess cytotoxicity, a total of 5 × 10 3 were plated in 96 well plates in DMEM with 10% FBS and 1% allowed adherence overnight.Then, cells were treated with increased concentrations of pharmaceutical agents and reader (Thermo Scientific, Finland), at 490 nm.Results were normalized with DMSO control values.To calculate IC 50 values the GraphPad Prism software (Version 9.0) was used to evaluate a nonlinear regression curve.All experiments were performed in triplicates at least three times.

Chromosome preparation and GTG-banding
The karyotype analysis was performed as described previously [18].HCB-541 cell line at the fifteenth passage was incubated with 120 ng/ml colcemid (Life Technologies-Gibco) for 125 min.The cells were harvested by treatment with 0.025% trypsin EDTA, suspended in KCl 0.075 M solution at room temperature for 5 min, and fixed with methanol/ acetic acid (3:1) five times.To examine the chromosomal distribution of constitutive heterochromatin, GTG-banding was performed using the standard trypsin/Giemsa method [19].The karyotype analysis of HCB-541 cells was done on twenty metaphases.

Mutational and fusion analysis and clinical significance variants classification
Mutations from the patient's FFPE tumor biopsy and HCB-541 cell line were evaluated by Next Generation Sequencing (NGS), using the Custom Cancer Solution cSTS kit (SOPHiA Genetics).This 48-gene panel plus microsatellite instability (MSI), includes all protein-coding exons for all transcripts of 8 genes, as well as a selection of protein-coding exons of 40 genes with ± 10 bp flanking into the intron and, if applicable, including 5' UTR exons that contain coding sequence (Supplementary Table 2).No normal DNA tissue (blood or adjacent normal skin) was available for germline mutation analysis.Genetic variants were evaluated in both HCB-541 cell line and the primary tumor.Moreover, the presence of gene rearrangements was performed on the HCB-541 cell line, using also the Custom Cancer Solution (cSTS) kit (SOPHiA GENETICS, Switzerland) according to the manufacturer's protocol.The somatic RNA panel for fusion research includes 16 genes: ALK, BRAF, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, MET exon 14 skipping, NTRK1, NTRK2, NTRK3, RET, ROS, ERG, PPARG and NRG1.
Briefly, from 20 ng of DNA, fragments were generated using an enzymatic fragmentation step.The three subsequent enzymatic steps, end-repair, A-tailing, and ligation to Illumina adapters, were performed to produce NGS libraries.A capture-based target enrichment was carried out on the pooled libraries.The quantitation of the final pool of libraries was performed using Qubit dsDNA HS fluorimetric assays (ThermoFisher).Quality control of fragment size was assessed using DNA ScreenTape analysis 4150 TapeStation system (Agilent).Sequencing was achieved with the final library concentration of 10 pM onto a 600-cycle format V3 flow-cell, via Illumina MiSeq platform Illumina.Data analysis was performed to detect Single Nucleotide Variants (SNVs), insertions/deletions (INDELs), and Copy Number Alterations (CNAs).Sequencing FASTQ data were analyzed by the Sophia DDM® platform (SOPHiA Genetics).For gene fusions, 200 ng of RNA was used to convert it into cDNA, and library construction followed the RNA target Oncology solution (ROS) kit by SOPHiA GENET-ICS, Switzerland, as per the manufacturer protocol.DNA fragments were generated through enzymatic steps, including end-repair, A-tailing, and ligation to Illumina adapters, to create NGS libraries.A capture-based target enrichment was applied to the pooled libraries.The final library pool was quantified using Qubit dsDNA HS fluorimetric assays from Life Technologies, USA.Quality control for fragment size was assessed using DNA ScreenTape analysis with the 4150 TapeStation.Data analysis was performed to detect fusion genes.Sequencing FASTQ data were analyzed by the Sophia DDM® platform (Sophia Genetics, Switzerland).
TERT hotspot promoter mutations were also validated by droplet digital PCR (ddpCR).To detected TERTp mutations was used 10 ng of DNA, 2X ddPCR Supermix for Probes (No dUTP) (Bio-Rad, Hercules, CA, USA), 5 M Betaine (Sigma), 80 mM EDTA (Sigma), 10U/ul CviQI enzyme (ThermoFisher) and the commercial assay TERT C228T: ddPCR™ Expert Design Assay: TERT C228T_113, Human, Homo sapiens (Bio Rad), following the manufacturer's protocol.Droplets were generated using a droplet generator (Bio-Rad).The PCR cycling parameters included 10 min at 95 °C, 50 cycles at 96 °C for 30 s and 61 °C for 30 s, and one cycle at 98 °C for 10 min followed by a 4 °C hold.Droplet fluorescence was assessed using a droplet reader (Bio-Rad).Analysis of the ddPCR data for allele calling and the calculation of absolute copy numbers were performed using QuantaSoft software, version 1.7.4 (Bio-Rad).

Molecular microsatellite instability (MSI)
MSI evaluation was also performed by multiplex PCR using 6 quasi-monomorphic mononucleotide repeat markers (BAT25, BAT26, NR21, NR24, NR27, HSP110) as described by Berardinelli et al. [22] PCR was performed using Qiagen Multiplex PCR Kit (Qiagen) with 50 ng of DNA and the following thermocycling conditions: 15 min at 95 °C; 40 cycles of 95 °C for 30 s, 55 °C for 90 s and 72 °C for 30 s; and a final extension at 72 °C for 40 min.PCR products were then submitted to capillary electrophoresis on an ABI 3500XL Genetic Analyzer (Ther-moFisher) according to the manufacturer's instructions.The results were analyzed using GeneMapper v4.1 software (ThermoFisher) to measure the fragment length in base pairs.The classification of instability is determined by the number of markers out of the quasi-monomorphic variation range (QMVR), being 2 or more markers classified as MSI-H (MSI-High), only one marker classified as MSI-L (MSI-Low) and cases without markers out of QMVR classified as MSS (Microsatellite stability), as previously reported [22].

Droplet digital PCR (ddPCR) detection for human papilloma virus (HPV)
The presence of HPV 16 and 18 was evaluated in the patient's tumor biopsy and HCB-541 cells, using ddPCR, as previously described [23].Briefly, for HPV detection, 10 ng of DNA were conducted to Singleplex PCR reactions with 2X ddPCR Supermix for Probes (No dUTP) (BioRad, Hercules, CA, USA), 900 nM of each primer and 300 nM of the probe in a total volume of 22 µL followed by droplet generation using an automated droplet generator (BioRad) according to the optimized protocol [23].Droplets were generated using a droplet generator (Bio-Rad).The PCR cycling parameters included 10 min at 95 °C, 50 cycles at 96 °C for 30 s and 61 °C for 30 s, and one cycle at 98 °C for 10 min followed by a 4 °C hold.Droplet fluorescence was assessed using a droplet reader (Bio-Rad).Analysis of the ddPCR data for allele calling and the calculation of absolute copy numbers were performed using QuantaSoft software, version 1.7.4 (Bio-Rad).Caski (+ HPV 16) and SiHa (-HPV) cell lines were used as controls and were obtained from the ATCC cell bank.

mRNA expression profiles by nanostring
mRNA expression of a panel of 770 genes associated with 13 cancer-associated canonical pathways related to basic cancer biology, was evaluated using the nCounter PanCancer Pathways Panel, NanoString technology (Nanostring Technologies, USA) for HCB-541 cells and HACAT (immortalized human keratinocyte cell line) cells (used as a non-tumor counterpart) for differential expression analysis, as previously reported [17].

Establishment of a primary cutaneous squamous cell carcinoma (cSCC) cell culture
A tumor biopsy in the region of the left armpit (Supplementary Fig. 1), was performed for anatomopathological analysis confirming the diagnosis of cutaneous squamous cell carcinoma (cSCC).Surgical specimens measuring 1.5 × 1.3 cm 2 , were collected by sterile scalpels in sterile tubes containing 5 mL of DMEM serum-free medium, supplemented with 1% penicillin-streptomycin.After cells disaggregate protocol two populations were observed, then over 15 days, showed a clustered cell group with fibroblastic morphology that were removed by differential trypsinizations steps to enrich the keratinocytes population.In the initial passages, the growth of the cell was observed in disconnected blotches, after several passages the cell lines acquired a similar keratinocytes behavior (Fig. 1A).Different growing conditions were done to analyze the HCB-541 growth rates by real-time protocol using xCEL-Ligence RTCA systems.We consider the culture growth rate doubling time between 19 and 22 h, exposed to 10% or superior FBS concentrations and greater than 24 h at concentrations less than 10% FBS concentrations, and without SFB, the HCB-541 cell line obtained a cell index of more than 50 h (Fig. 1B).
HPV infection was not detected in both patient's tumor and HCB-541 cell line (Supplementary Fig. 2).

Cell line characterization by immunohistochemistry and immunocytochemistry
Hematoxylin and eosin staining of the tumor biopsy and HCB-541 cell line showed similar profile, with clear cytoplasm and large, basophilic nuclei with visible nucleoli and a variable number of mitoses (Fig. 1C).HCB-541 cell depicted a weak vimentin staining, strong staining of Ki67, cytokeratin 20, cytokeratin A1/A3, cytokeratin 17 and cytokeratin 8 markers detected in early and late passages (Fig. 1C).A similar expression profile was observed on HACAT (immortalized human keratinocytes) and A431 (epidermoid carcinoma cell line) (Fig. 1C).
Positive immunostaining for the CK5/6, p63, p40 and CD10 antibodies was exclusively evident in the tumor biopsy and xenograft model (Fig. 2A).We observed that only CD10 positivity was conserved in the HCB-541 cell line and also in the xenograft model (Fig. 2A).Positive p63 immunohistochemical staining was exclusively discerned within the tumor biopsy and xenograft model (Fig. 2A).

Cell line authentication and mycoplasma contamination test
The HCB-541 cell line was mycoplasma free.Short tandem repeat fingerprinting was performed using DNA from the cell line and tumoral biopsy.We detected the loss of one allele from D5S818 and TPOX regions after the establishment of the culture, and the HCB-541 cell line showed at least 90% identical short tandem repeats compared with patient's biopsy (Supplementary Table 3).

In vivo tumorigenicity of HCB-541
The immunohistochemistry analysis indicated that the xenografts replicated the histopathological features of the cSCC tumor biopsy (Fig. 2A).The tumorigenic potential of HCB-541 was investigated in vivo, by subcutaneously injection of 1.5 × 10 6 or 3 × 10 6 cells, into the right flank of female NOD Scid Gamma (NSG) mice and we observed tumor growth after 15 days in all animals tested (Fig. 2B, C).In 15 days, the weight of tumors varied from 0.1 to 0.25 g for mice injected with 1.5 × 10 6 cells and 0.4-0.5 g for mice injected with 3 × 10 6 cells (Fig. 2C).
According to International System for Human Cytogenomic Nomenclature, the representative karyotype was 40

Mutational analysis
Mutation analysis in both patient´s tumor biopsy and cell line revealed 11 variants commun (Table 1).Among the variants identified the ones at HRAS p.(Gln61His), TERT (1295228 C > T (termed TERT C228T)), and TP53 p.(Arg248Leu), were classified as pathogenic or likelypathogenic.We also found mutation only in the tumor tissue in the genes DICER, ESR1, KIT, PDGFRA, and RAF1 (Table 1).Finally, one variant at NOTCH1 was detected only in the cell line.(Table 1).The hotspot TERT promoter mutation (C228T), was further confirmed by ddPCR in both patient's tumor biopsy and in the HCB-541 cell line (Fig. 3 A, B).
Any gene fusion was identified in the HCB-541 cell line.
Of notice, the MSI analysis revealed that both the HCB-541 cell line and primary tumor tissue presented alteration of BAT26, NR27, BAT25, and NR24 markers, leading to an MSI-High phenotype (Supplementary Fig. 4).

mRNA expression analysis
We detected the mRNA expression profile of cancer-related genes of the HCB-541 cell line and compared it with the HACAT (non-tumor skin keratinocyte) profile.We found 37 genes differentially expressed in HCB-541 (Fig. 3C and supplementary Table 4).The most remarkable fold change was the overexpression of ITGB3, CSF2, LAMA1, ETV1, ZIC2, FLNC, and VEGFC genes in HCB-541 compared with HACAT.At variance, BMP7, WNT10A, IGFBP3, CEBPA, The in silico interaction network analysis using these 11 upregulated genes in the HCB-541 cells showed a tight network of PPI (protein-protein interaction) pathway enrichment (p = 0.00497) and functional enrichments suggesting interaction with viral myocarditis (red color), focal adhesion (blue color), VEGF signaling (green color), fluid shear stress and atherosclerosis (yellow color) and AGE-RAGE signaling pathway in diabetic complications process (pink color) (Fig. 3D).The same in silico interaction network analysis of the 26 downregulated genes PPI enrichment of p-value of 1.0e-16 and functional enrichments suggest interaction with Notch (red color), p53 signaling (blue color), Basal cell carcinoma (green color), Bladder cancer (yellow color) and ECM-receptor interactions (pink color) (Fig. 3F).
EGFR protein expression was associated with the passage number, in which we detected lower phosphor-EGFR protein levels in higher passages, compared with long culture time (> 20 passage).Contrarily, FGFR1 and ROR2 protein expression levels were higher in the late passage.Among the tested receptors, ROR2 showed higher levels of phosphorylation (Fig. 4C).MAPK protein expression showed several MAPK phosphorylated protein expressions such as AKT1, ERK1/2, GSK-3, HSP27, JNK2, MKK3, P53 and RSK1 (Fig. 4D).We detected a discrete ERK decrease in the late passage compare with initial, and HCB-541 cell line after 20 passages (Fig. 4E, F).A431 cell line was used as a model human cell line of epidermoid carcinoma.

Discussion
Advanced cutaneous squamous cell carcinoma (cSCC) is a challenging tumor type that does not respond to surgery or radiotherapy and represents a rare subgroup of cSCC.Currently, only one cSCC cell line (A431) is commercially available, and few studies described metastatic cSCC lines (UT-SCC-7, 59A, and 115), but their molecular profiles have not been reported [24,25].In the present study, we established a novel cSCC cell line, HCB-541, from a 67-year-old male patient with advanced squamous cell carcinoma in the armpit.
We used a combination protocol connecting physical and enzymatic tissue digestion to establish the HCB-541 cell line.A recent study reported three decades of work, leading to a panel of cutaneous SCC cell lines [26].The authors described 16 cell lines derived from 11 patients and reported a long-time interval (10 to 30 years) for the development of these cell lines.Our cSCC cell line, HCB-541, showed a very fast growth, and the formation of tumor in xenograft only after 15 days of cell injection into the mice, suggesting a very aggressive behavior of the HCB-541 cell line.
We found that in supplemented conditions of 5 or 10% FBS, the HCB-541 cell line displayed a doubling time of about 19-22 h, respectively, similar to the 18-24 h doubling time of A431 cells [27].The establishment of primary cell cultures is troublesome and time-consuming, and in addition, it has high fibroblastic and microbiologic contamination rates [28].We eliminated fibroblastic contamination from HCB-541 by using a fast trypsinization protocol after higher passage.After fibroblastic exclusion, the cytokeratin profile showed a stronger immunostaining for pan-cytokeratin A1/A2, and Ki67, which are present in epithelial cells and carcinomas [29].Interestingly, the HCB-541 cell line had positive staining for cytokeratin 20, which is commonly used for the diagnosis of Merkel cell carcinoma [30] and metastatic salivary gland tumors [31].After the cell line establishment, only CD10 immunostaining was preserved in the HCB-541 cell line.Some studies have already associated CD10 overexpression with other tumor types, such as SCC [32], cutaneous basal cell carcinoma (BCC) [33].
The karyotype analysis was conducted on the HCB-541 cell line after the twentieth passage.Specifically, 12 out of 20 metaphases exhibited a hypodiploid karyotype, while the remaining 8 out of 20 displayed a hypotetraploid karyotype.The karyotype of HCB-541 cells showed heterogeneous chromosomal populations, which is common in solid tumors [34].The identification of a hyperdiploid karyotype, characterized by its complexity and the presence of multiple cell populations, has been documented in various primary cell culture establishment, including breast tumor cell lines [35] and prostate models [36].This phenomenon is attributed to the emergence of cell subclones throughout the tumor's evolutionary process.To enhance our understanding, future investigations employing single-cell RNA sequencing analysis on the HCB-541 cell line will be performed for more precise insights into its cellular heterogeneity.
Our mutation analysis identified the presence of mutations in several cancer-related genes previously reported to be important in cutaneous squamous cell carcinoma [9,37].The pathogenic TP53 variant p.(Arg248Leu) was observed in both tumor tissue and the HCB-541 cell line.This variant is considered highly pathogenic and has been reported in 16 morphological tumor types, including squamous cell carcinoma, glioblastoma, adenocarcinoma, hepatocellular carcinoma, and transitional cell carcinoma [38].Interestingly, this variant has not been reported in metastatic cSCC before.We also the hotspot detected the promoter TERT C228T mutation in both the patient's metastatic tumor and HCB-541 cell line by NGS panel and further confirmed by droplet digital PCR (ddPCR).This mutation has been associated with a poorer prognosis and increased risk of lymph node metastasis in cSCC patients [37]).However, only a few studies have analyzed cSCC and revealed rates varying from 34.7% in invasive stages to no more than 19.4% in in situ cSCC [39].Another important mutated oncogene identified was HRAS, with the presence of a p.(Gln61His).This HRAS variant is reported to be mutated in this tumor type and is associated with increased proliferation upon Vemurafenib exposure [40].Due to the lack of normal tissue, we were unable to confirm the somatic nature of the mutations identified, yet several studies suggest that these oncogenic alterations are somatic [41,42].
We did not find any gene rearrangement in the HCB-541 cell line.This is in line with the literature that shows similar observation, with the exception of the ADCK4-NUMBL recurrent gene fusion, which is not present in our NGS assay [43].Importantly, we found that both the tumor and HCB-541 cell line exhibited an MSI phenotype.The data on MSI is less explored and conflicting.Some studies reported that MSI is present in a small subset of cases (~ 2%) with invasive or metastatic behavior [44].This finding is very relevant since it is well-known that MSI is an agnostic biomarker of immunotherapy response [45].
Infection with human papillomavirus (HPV) of the beta genus (betaHPV), which includes subtypes 16 and 18 considered high-risk, is seldom linked to the onset of cutaneous squamous cell carcinoma (cSCC).However, it can contribute to skin cancer development in specific cases, such as patients with the genetic disorder Epidermodysplasia verruciformis (EV) or those who are immunocompromised [46].Since the association between beta HPV and cutaneous SCC remains unclear [47], we analyzed its presence in the HCB-541 cell line and observed the absence of subtypes 16 and 18.
Protein enrichment pathway analysis revealed a strong correlation with other disorders, such as viral myocarditis, atherosclerosis, and diabetic complications.Interestingly, the patient had a history of two myocardial infarctions prior to the development of cutaneous squamous carcinoma.Downregulated genes in HCB-541 were associated with complex network pathways, such as Notch, which has been implicated in the carcinogenesis process of squamous epithelial malignancies [48].The tyrosine kinase receptor ROR2 has been reported as upregulated gene in numerous types of human cancer, such as laryngeal squamous cell carcinoma, and endometrial cancer [49,50].For the first time, we observed higher expression of the tyrosine kinase receptor ROR2 in a metastatic cutaneous squamous carcinoma cell line.
Our investigation has unveiled significant therapeutic responses with second-generation EGFR inhibitors (afatinib and allitinib), which selectively target multiple members of the ErbB receptor family.We hypothesize that the efficiency of these inhibitors was achieved by irreversible binding to the tyrosine kinase receptor, as compared to the reversible action promoted by erlotinib and cetuximab agents.HCB-541cell line was also sensitive to DNA alkylating agents.
Concluding, we have established an immortalyzed metastatic cutaneous squamous cell carcinoma (cSCC) cell line, HCB-541, which carries pathogenic mutations of TP53, HRAS and TERT and shows an MSI-High status.The high tumorigenicity of this cell line is also associated with the overexpression of several tumor markers, including ROR2 and FGFR, and it showed sensitivity to irreversible pan-ErbB inhibitors and DNA alkylating agents.The HCB-541 can constitute a useful preclinical model for investigating cSCC biology and its response to therapeutic targets.

Fig. 1
Fig. 1 Morphological and proliferative characterization of HCB-541 cell line.A Representative photomicrograph of the HCB-541 cell line at 10 × magnification, showing the establishment days.B Realtime cell analysis showing the doubling time of HCB-541 cells under different SFB concentrations, with bars representing time (hours) at 60 h.C Hematoxylin eosin staining and immunocytochemistry of

Fig. 3
Fig. 3 TERT promoter mutation analysis, and gene expression analysis of a pan-cancer panel by NanoString™ and functional pathways analysis A TERT promoter gene analysis from patient's tumor.B TERT promoter gene analysis from HCB-541 tumor cell line after establishment.C Heatmap of genes altered in HCB-541 compared

Fig. 4
Fig. 4 Protein profile of MAPK and RTK phosphorylated of HCB-541 cell line and sensibility to different antineoplastic agents.A RTK phosphorylated array of HCB-541 cell line; B Densitometric analyses of RTK detection array are represented by bar graphs; C RTK phosphorylated profile of HCB-541 by Western blot.D MAPK phosphorylated array of HCB-541 cell line.E Densitometric analyses of MAPK detection array are represented by bar graphs.F MAPK phosphorylated profile of HCB-541 by Western blot analysis.Each RTK and MAPK is duplicate in the arrays (two spots side by side),

Table 1
Mutation profile of both tumor biopsy and HCB-541 cell line Tier II Potential Clinical Significance, Tier III Unknown Clinical Significance, Tier IV Benign or Likely Benign; SNV Single Nucleotide Variant,